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Hi,
You can try to get a mass spectra of your protein with the metal ie
your crystal, and another mass spectra of your protein, where it is
denatured to loose its metal. The difference in the mass between the
two spectra , may reflects the identity of the metal bound to your
protein.
-Selvaraj
>
> Hello-
> Thanks for entertaining my non-ccp4 question...
>
> I am refining a crystal structure at ~2 Angstroms resolution in which I
> find a large, unexpected electron density that is potentially a Zinc
> ion because it is coordinated by 2 Histidines, 1 Glutamic acid, and
> possibly a water molecule.
>
> This site appears as a strong peak in anomalous difference Patterson
> maps (the data was collected at SelMet wavelength 0.979 Angstroms and I
> believe that Zn's absorption edge occurs around 1.2 Angstroms). The
> problem is, I didn't add any zinc in any purification or
> crystallization conditions. I DID affinity purify the protein with
> nickel beads, so potentially it could be a Nickel ion. I should also
> say that during refinement in refmac where my Zn occupancy is held at
> 1, that I obtain a negative peak in the 2Fo-Fc in this position, but if
> I leave the site unoccupied- I get a huge positive peak- so I either
> have the wrong species defined or my occupancy is not 100%- right?
>
> Is there any way short of biochemical means (ITC, mutation, etc.) to
> figure out what species is occupying this electron density? I have
> thought of atomic absorption- has anyone tried it to determine the
> metal species in a protein? Does it seem possible that I could have a
> Zn ion in my protein crystal where the Zn could only come from our
> standard DI water supply?
>
> Thanks so much!
> Wendy Ryan Gordon
>
